The archeological register of Patagonia is extremely interesting; it has the oldest record of human occupation in America (Monte Verde, 14,500 BP). It also has many Holocene sites that contain human bones as old as 8,800 years BP. Due to the favorable environmental conditions (cold and dry), the conservation of human remains is good enough to allow obtaining mitochondrial DNA, and in exceptional cases, nuclear DNA. We extracted and amplified mitochondrial DNA sequences of samples from northern, central and southern Patagonia distributed in a temporal range from 8,800 to 400 years AP. The majority of the samples studied belonged to haplogroups C and D; in one proto- Kaweskar we found haplotype C (16318G), previously described in a historical Aonikenk. Haplotype D4h3 (16241G and 16342C), which is found in present Patagonian populations and was recently described in a skeleton of 10,300 years BP in Alaska was also found, reinforcing the hypothesis of a connection between the populations of Patagonia and the hunter-gatherers of the beginning of the Holocene. In northern Patagonia we also detected the presence of haplogroup B in two individuals from the early Holocene. This haplogroup had not been found in present-day populations south of 43 degrees south latitude. Finally, in the most recent samples (400 years BP) we found haplotype D (16187T), very common in current populations of southern Chile, suggesting a possible population movement between them.